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Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Gibberellins inArabidopsis

Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 47, Issue of November 22, pp. 45188 –45194, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Gibberellins in Arabidopsis* Received for publication, August 23, 2002, and in revised form, September 12, 2002 Published, JBC Papers in Press, September 12, 2002, DOI 10.1074/jbc.M208659200 Hiroyuki Kasahara‡, Atsushi Hanada‡, Tomohisa Kuzuyama§, Motoki Takagi§, Yuji Kamiya‡ and Shinjiro Yamaguchi‡ From the ‡Laboratory for Cellular Growth and Development, Growth Physiology Research Group, Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan and §Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan already implicated in the 1960s from feeding experiments with Gibberellins (GAs) are diterpene plant hormones es- 14 14 sential for many developmental processes. Although the CO and [ C]MVA, which showed that these substrates GA biosynthesis pathway has been well studied, our labeled distinct groups of terpenoids (5). The MEP pathway knowledge on its early stage is still limited. There are was first described for eubacteria (6), and enzymes catalyzing two possible routes for the biosynthesis of isoprenoids the MEP pathway have been identified mainly in Escherichia leading to GAs, the mevalonate (MVA) pathway in the coli (7, 8). Although the precise reactions in the late steps of cytosol and the methylerythritol phosphate (MEP) path- MEP pathway have still to be determined, the overall pathway way in plastids. To distinguish these possibilities, in E. coli has recently been proposed (9). Because orthologs of metabolites from each isoprenoid pathway were selec- each of the bacterial genes of the MEP pathway are present in tively labeled with Cin Arabidopsis seedlings. Arabidopsis thaliana (8, 10), the same set of enzymes are likely Efficient C-labeling was achieved by blocking the to be involved in the MEP pathway in plants. endogenous pathway chemically or genetically during Gibberellins (GAs) are a class of plant hormones essential for the feed of a C-labeled precursor specific to the MVA or many aspects of plant growth and development, such as seed MEP pathways. Gas chromatography-mass spectrome- germination, stem elongation, and flower development (11, 12). try analyses demonstrated that both MVA and MEP The GA biosynthesis pathway in higher plants has been stud- pathways can contribute to the biosyntheses of GAs and ied in detail in cell-free systems from immature seeds of campesterol, a cytosolic sterol, in Arabidopsis seedlings. Cucurbita maxima, Pisum sativum, and Phaseolus vulgaris While GAs are predominantly synthesized through the (13). Because C-labeled MVA was efficiently incorporated MEP pathway, the MVA pathway plays a major role in into ent-kaurene (a GA precursor) in these cell-free systems, it the biosynthesis of campesterol. Consistent with some has long been assumed that GAs are derived from MVA in crossover between the two pathways, phenotypic de- fects caused by the block of the MVA and MEP pathways plants. The GA biosynthesis pathway has also been studied were partially rescued by exogenous application of the extensively in a GA-producing fungus Gibberella fujikuroi (14). MEP and MVA precursors, respectively. We also provide The incorporation of labeled MVA into GAs in cultured mycelia evidence to suggest that the MVA pathway still contrib- of this fungus also supported the premise that GAs are formed utes to GA biosynthesis when this pathway is limiting. from MVA in this organism (15). Several lines of evidence from recent work have indicated that ent-kaurene is synthesized in the plastids of plants (16 – Isoprenoids comprise a broad range of natural products that 19). Therefore, the MEP pathway in plastids may play a role in are synthesized by the condensation of the two precursors, providing IPP and DMAPP for ent-kaurene biosynthesis (Fig. isopentenyl diphosphate (IPP) and dimethylallyl diphosphate 1). There is some indirect evidence to support this hypothesis. (DMAPP) (1). Plants have two distinct biosynthetic routes for Antisense suppression of genes encoding enzymes in the Ara- the formation of IPP and DMAPP, the mevalonate (MVA) path- bidopsis MEP pathway resulted in elevated expression of a way and the newly discovered methylerythritol phosphate GA-down-regulated gene, GA4 (20), and reduced production of (MEP) pathway (Fig. 1) (2, 3). In plants, the MVA pathway ent-kaurene (21). Recently, the biosynthetic origin of stevio- plays an essential role in the biosynthesis of sterols and ses- side, of which the aglycone is a derivative of ent-kaurenoic acid, quiterpenoids in the cytoplasm (4). On the other hand, the MEP has been studied in Stevia rebaudiana leaves (22), where this pathway is generally responsible for the formation of carote- diterpene glycoside accumulates to more than 10% of the leaf noids, mono- and di-terpenoids, plastoquinones, and the prenyl dry weight as a secondary metabolite. This high abundance 13 13 group of chlorophylls in plastids (3). The occurrence of two allowed the C-labeling pattern from [1- C]glucose in steviol separate isoprenoid biosynthesis pathways in plants was to be determined by NMR and indicated that steviosides are synthesized through the MEP pathway. However, this method * The costs of publication of this article were defrayed in part by the is not feasible for GAs due to their low abundance in plant payment of page charges. This article must therefore be hereby marked tissues. Thus, to determine whether GAs are synthesized “advertisement” in accordance with 18 U.S.C. Section 1734 solely to through the MEP pathway in general, a more sensitive system indicate this fact. for detecting the labeled products is required. To whom correspondence should be addressed. Tel.: 81-48-467- 9552; Fax: 81-48-462-4691; E-mail: [email protected]. To this end, we used an Arabidopsis albino mutant cla11, The abbreviations used are: IPP, isopentenyl diphosphate; GA, gib- which is defective in 1-deoxy-D-xylulose 5-phosphate synthase berellin; DMAPP, dimethylallyl diphosphate; DX, 1-deoxy-D-xylulose; in the MEP pathway (23). The cla11 phenotype can be res- MEP, methylerythritol phosphate; MVA, mevalonate; MVL, mevalono- cued almost completely by treatment with exogenous 1-deoxy- lactone; TMS, tetramethylsilane; GC-MS, gas chromatography-mass spectrometry; MS medium, Murashige and Skoog medium. D-xylulose (DX), which is converted to a MEP pathway inter- 45188 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Contribution of the MVA and MEP Pathways to GA Biosynthesis 45189 FIG.1. The two possible isoprenoid biosynthesis pathways leading to GAs in plants. A cell-free system from C. maxima endosperm can convert MVA to ent-kaurene and GAs. ent-Kaurene is pro- duced in plastids and then converted to GAs by following oxidation reactions in cytosol. Crossover of common isoprenoid precursors (IPP, geranylgeranyl diphos- phate (GGPP), or farnesyl diphosphate (FPP)) between cytosol and plastids has been suggested in several plant species. Dashed arrows indicate multiple biosyn- thetic steps. HMGR, 3-hydroxy-3-meth- ylglutaryl-CoA reductase; DXS, 1-deoxy- D-xylulose 5-phosphate synthase; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reduc- toisomerase; GAP, glyceraldehyde 3-phosphate. concentration 1 M) was added, and then the plants were grown for an mediate 1-deoxy-D-xylulose 5-phosphate in plants. This system additional 3 days before analyzing ent-kaurene. GA and campesterol allowed us to label the products from the MEP pathway effi- were analyzed without uniconazole treatment. ciently in vivo using [2- C]DX. To evaluate the role of the Feeding of [2- C]MVL to Mevastatin-treated Plants—Wild-type cytosolic MVA pathway in GA biosynthesis in the same system, seedlings were grown for 5 days on MS agar media before transferring C-labeled mevalonolactone (MVL) was fed to plants that were to MS liquid media. Immediately after the transfer, mevastatin (MeOH treated with mevastatin, an inhibitor of the MVA pathway. solution, final concentration 10 M) and [2- C]MVL (filter-sterilized H O solution) were added aseptically to the liquid media, and the plants Our gas chromatography-mass spectrometry (GC-MS) analysis 2 were grown for 9 days. ent-Kaurene, GA , and campesterol were ana- demonstrated that GAs are predominantly synthesized from lyzed as described above. the MEP pathway in Arabidopsis seedlings. However, our GC-MS Analysis of ent-Kaurene, GA , and Campesterol—For ent- results also indicated a minor contribution of the MVA pathway kaurene analyses, seedlings (3 g) were pulverized with a mortar and to GA biosynthesis. Cooperation of both isoprenoid pathways pestle chilled by liquid N . Powdered tissues were extracted with 80% was also evident for the biosynthesis of the sterol campesterol MeOH (25 ml) overnight. The 80% MeOH extract was then partitioned against n-hexane (15 ml) three times, and the combined n-hexane (a precursor for brassinosteroids), which is formed in the fraction was evaporated to 1 ml. The n-hexane fraction was subjected to cytosol. SiO gel column chromatography (column size, 5  2 cm) and eluted with 15 ml of n-hexane. The eluate was carefully evaporated to 20 l EXPERIMENTAL PROCEDURES under gentle N flow and analyzed by GC-MS. GC-MS analysis was Plant Materials and Growth Conditions—A. thaliana ecotype Was- performed on a GC-mate II mass spectrometer (JEOL, Tokyo, Japan) silewskija (WS) was used in this study. Plants were grown at 21 °C connected to a Agilent 6890 series GC system with a 30-m  0.25-mm using a 16-h light/8-h dark photoperiod with cool-white illumination. capillary column DB-5 MS (0.25-m film thickness, J & W Scientific). Wild-type (WS-2) and the cla11 mutant (24) were germinated and GA and campesterol were extracted from 2227-g and 0.4-g seedlings grown on Murashige and Skoog (MS) agar media (pH 5.7) supplemented and derivatized to methyl ester and TMS ether, respectively, and ana- 1 1 with thiamin hydrochloride (3 gml ), nicotinic acid (5 gml ), pyr- lyzed as reported previously (29, 30). ent-Kaurene and GA were iden- idoxin hydrochloride (0.5 gml ), and 1 or 3% (w/v) sucrose. Liquid tified by Kovats retention indices (31), and full mass spectra obtained culture was carried out in 15 ml of MS media in 100-ml flasks on a by GC-MS (29, 32). shaker (100 rpm). Chemicals—DX and [2- C]DX (95% labeled) were synthesized using RESULTS pyruvate or [2- C]pyruvate (99% labeled, Aldrich) as previously re- 13 13 13 ported (25). To determine the C-labeling ratio of [2- C]DX, DX and Incorporation of [2- C]DX into ent-Kaurene—The Arabidop- [2- C]DX (ca. 100 ng) were converted to trimethylsilyl derivatives by sis cla11 mutant is defective in 1-deoxy-D-xylulose 5-phos- heating at 70 °C with N,O-bis(trimethylsilyl)acetamide  trimethyl- phate synthase in the MEP pathway and displays a seedling- chlorosilane  trimethylsilylimidazole (3:2:3, 50 l, Supelco) and pyri- lethal albino phenotype (23, 24). Previous studies show that the dine (50 l) for 20 min and then analyzed by GC-MS as previously 13 cla11 phenotype is in part restored when grown on agar reported (26). DL-MVL and [2- C]MVL (99% labeled) were purchased media containing DX (23). We found that the cla11 phenotype from Aldrich, and mevastatin was from Sigma. ent-[1,7,12,18- C ] Kaurene was produced from [2- C]MVA by a cell-free system prepared can be better rescued in liquid culture than on agar media in from C. maxima endosperm as previously reported (27, 28). the presence of DX, possibly because of better uptake of the Feeding of [2- C]DX to the cla1-1 Mutant—The albino cla11 ho- chemical by seedlings. Fig. 2A shows that, in the presence of mozygotes were selected from the progeny of CLA1/cla11 plants after 0.8 –1.0 mM DX, the phenotype of the cla11 plants was re- incubation on MS agar media for 9 days. The cla11 seedlings were 13 stored to that of the wild type. To label isoprenoids that are then transferred to MS liquid media. [2- C]DX was dissolved in H O, produced via the MEP pathway in vivo, the cla11 mutant was filter-sterilized, and added aseptically to the liquid culture. Twelve days after the transfer to liquid media, uniconazole (EtOH solution, final treated with 1 mM [2- C]DX for 15 days. Again, the rescue of 45190 Contribution of the MVA and MEP Pathways to GA Biosynthesis FIG.2. A–D, effect of DX and MVL on Arabidopsis cla11 and mevastatin-treated wild-type seedlings. Numbers indicate concentra- tions (mM)ofDX(in A and D) or MVL (in B and C). All experiments were performed in MS liquid media supplemented with 1% sucrose. A, restoration of cla11 seedlings by DX. The albino cla11 seedlings (9 days after sowing) were incubated in MS liquid media for 10 days with DX. For wild-type, see Control in B. B, restoration of mevastatin-treated wild-type seedlings by MVL. WT seedlings (5 days after sowing) were incubated for 7 days in MS liquid media with MVL in the presence of mevastatin (10 M). Control indicates the plant incubated without mevastatin and MVL. C, effect of MVL on cla11 seedlings. The albino cla11 seedlings (5 days after sowing) were incubated in MS liquid media for 3 days with MVL. D, effect of DX on mevastatin-treated wild-type seedlings. WT seedlings (5 days after sowing) were incubated for 5 days in MS liquid media with DX in the presence of mevastatin (10 M). Control indicates the plant incubated without mevastatin and DX. the albino phenotype nearly to wild-type confirmed that addition to the molecular ion at m/z 272, four isotope peaks at [2- C]DX was metabolized as required. m/z 273, 274, 275, and 276 were observed, indicating that 1– 4 To examine the role of the MEP pathway in GA biosynthesis, C labels were introduced into ent-kaurene (Table I). Subtrac- 13 13 13 we first determined the incorporation of [2- C]DX into ent- tion of natural C abundance indicated that [2- C]MVL pro- kaurene, a tetracyclic hydrocarbon precursor for all GAs, by vided 53% of the isoprene units to ent-kaurene under this GC-MS. Because ent-kaurene accumulates at low levels in Ara- condition. As discussed above, the mass spectrum of ent-kau- bidopsis seedlings (data not shown), plants were treated with 1 rene produced from [2- C]MVA through the MVA pathway M uniconazole for 3 days to block ent-kaurene metabolism contains a fragment ion at m/z 233 ([M-43] ), because ring D of before GC-MS analysis (21). If ent-kaurene is synthesized from ent-kaurene would not be labeled with C. Considering the 13 13 13 [2- C]DX through the MEP pathway, four C atoms would be amount of C incorporation (53%) calculated from the molec- introduced at the positions shown in Fig. 3 (Route 2). Consist- ular ion cluster, the relative intensity of the fragment ion at ent with this prediction, the mass spectrum of ent-kaurene m/z 233 confirms the incorporation of [2- C]MVL into ent- from the [2- C]DX-treated cla11 plants indicated a peak at kaurene through the MVA pathway. These results illustrate m/z 276, which corresponds to the molecular ion with four C that the MVA pathway also contributes to the biosynthesis of atoms per molecule (Table I). The peak at m/z 229 [M-43] of ent-kaurene in Arabidopsis seedlings. 13 13 non-labeled ent-kaurene is a fragment ion after loss of ring D Incorporation of [2- C]DX and [2- C]MVL into Campes- (C H and three hydrogen atoms) (Fig. 3), as previously dem- terol—Our feeding experiments showed that both the MEP and 3 4 onstrated by deuterium-labeling experiments (32). Impor- MVA pathways can provide precursors for the biosynthesis of tantly, the corresponding ion peak from the [2- C]DX-treat- ent-kaurene. To study the contributions of these two pathways ed plants was observed at m/z 232 ([M-44] ). This indicates to cytosolic sterol biosynthesis in Arabidopsis seedlings, the 13 13 13 the loss of one C label in ring D and is consistent with the incorporation of [2- C]DX and [2- C]MVL into campesterol predicted labeling pattern through the MEP pathway from was analyzed by GC-MS. [2- C]DX. To confirm this result, we analyzed ent-kaurene The mass spectrum of campesterol-TMS from the 13 13 produced from [2- C]MVA by a cell-free system from C. [2- C]MVL-fed seedlings showed that 98% of its isoprene units maxima endosperm (27, 28), where ring D would not contain was C-labeled (Table I). A peak at m/z 477, which corresponds 13 13 13 C-labels (Fig. 3, Route 3). GC-MS showed that four C to the molecular ion with five C atoms per molecule, is in atoms per molecule were incorporated into ent-kaurene and agreement with the expected labeling pattern because one of that the corresponding fragment ion was at m/z 233 ([M- the six C-labels introduced in the precursor squalene will be 43] ). Subtraction of natural C abundance revealed that eliminated by C-4 demethylation during campesterol biosyn- 99% of the C building blocks had been derived from thesis (Fig. 3, Route 4). The fragment ion peak at m/z 343 [2- C]DX through the MEP pathway in the cla11 seedling ([M-129] ) of non-labeled campesterol-TMS is attributed to loss (33). of C-1, -2, and -3 and a TMS-ether group of the A-ring (35), as Incorporation of [2- C]MVL into ent-Kaurene—Mevastatin shown in Fig. 3. The corresponding fragment ion from the inhibits 3-hydroxy-3-methylglutaryl-CoA reductase in the [2- C]MVL-fed seedlings at m/z 347 ([M-130] ), which indi- MVA pathway and causes severe growth inhibition, which is cates loss of one C-label, is consistent with the predicted restored by simultaneous application of MVL. To study the labeling pattern (Fig. 3). involvement of the MVA pathway in GA biosynthesis, To examine the role of MEP pathway in cytosolic sterol 13 13 [2- C]MVL was fed to Arabidopsis seedlings that were incu- synthesis, campesterol was analyzed in the [2- C]DX-treated bated with mevastatin to label MVA-derived isoprenoids (34). In cla11 seedlings by GC-MS. Campesterol can be labeled with 13 13 our liquid culture conditions, 10 M mevastatin was effective in C at six positions when [3- C]IPP, originating from inhibiting seedling growth, and this inhibitory effect was nearly [2- C]DX, is incorporated (Fig. 3, Route 1), in contrast to the 13 13 completely abolished by the addition of 3 mM MVL (Fig. 2B). five C atoms incorporated from [2- C]MVL. The mass spec- ent-Kaurene was analyzed by GC-MS from seedlings that trum of campesterol-TMS from [2- C]DX-fed cla11 seed- were treated with 10 M mevastatin and 3 mM [2- C]MVL. In lings showed a peak at m/z 478 (Table I). This indicated that Contribution of the MVA and MEP Pathways to GA Biosynthesis 45191 13 13 FIG.3. Predicted labeling patterns of ent-kaurene, GA and campesterol with [2- C]DX or [2- C]MVL. Red arrows indicate possible 13 13 metabolic conversion of [2- C]DX through the MEP pathway, and blue arrows are those of [2- C]MVL through the MVA pathway. Red stars 13 13 13 specify positions of C atoms from [2- C]DX, and blue stars specify those from [2- C]MVL. The ring D of ent-kaurene is shown in orange, the A-ring of campesterol was shown in pink. , derivatized from campesterol by trimethylsilylation with N-methyl-N-trimethylsilyltrifluoroacetamide. , derivatized from GA by methylation using diazomethane. DXP, 1-deoxy-D-xylulose 5-phosphate; GGPP, geranylgeranyl diphosphate; or FPP, farnesyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A. TABLE I GC-MS data for ent-kaurene and campesterol-TMS Feeding experiments were carried out in MS liquid media supplemented with 3% sucrose (total 15 days of incubation). KRI, Kovats retention index. Mass spectrum KRI % Incorporation Molecular ion Fragment ion % relative intensity ent-Kaurene Standard 2082 272 (100) 273 (21) 274 (3) 229 (93) 230 (18) 231 (4) 13 b [2- C]DX/cla1–1 2082 275 (25) 276 (98) 277 (21) 232 (100) 233 (22) 234 (3) 99 13 c [2- C]MVL/mevastatin 2082 272 (70) 273 (48) 274 (52) 27 (99) 229 (78) 230 (64) 231 (78) 232 (100) 53 276 (71) 233 (49) [2- C]MVA/C. maxima 2082 276 (100) 277 (15) 232 (39) 233 (100) Campesterol TMS Standard 472 (42) 473 (16) 474 (4) 343 (100) 344 (27) 345 (4) 13 b [2- C]DX/cla1–1 472 (63) 473 (65) 474 (42) 47 (23) 343 (88) 344 (100) 345 (89) 346 (60) 27 476 (15) 477 (13) 478 (13) 347 (40) 348 (34) 349 (33) 350 (7) 13 c [2- C]MVL/mevastatin 476 (6) 477 (38) 478 (14) 345 (12) 346 (43) 347 (100) 348 (26) 98 The intensity of base ion peak was set as 100%. b 13 The cla1–1 seedlings were fed with 1 mM [2- C]DX. c 13 The wild-type seedlings were fed with 3 mM [2- C]MVL in the presence of mevastatin. a maximum of six C atoms were incorporated per molecule, MVA pathway also contributes to the biosynthesis of campes- which is consistent with the incorporation of IPP/DMAPP terol in Arabidopsis seedlings. 13 13 13 derived from [2- C]DX into cytosolic campesterol biosynthe- Incorporation of [2- C]DX and [2- C]MVL into GA —Al- sis. Furthermore, considering the amount of C incorpora- though ent-kaurene is a common intermediate for all GAs, it is tion (27%) estimated from the molecular ion cluster, the also known to serve as a precursor for other diterpenoids such relative abundance of the fragment ion at m/z 349 indicates as stevioside in S. rebaudiana (22) and kaurenolides in C. no C label at C-1 of the A-ring. This observation agrees with maxima (36, 37). In Arabidopsis, it has not been established the expected labeling pattern through the MEP pathway whether ent-kaurene serves as a precursor solely for GAs. To from [2- C]DX (Route 1). These results indicate that the determine conclusively the role of MEP pathway in the biosyn- 45192 Contribution of the MVA and MEP Pathways to GA Biosynthesis TABLE II 13 13 Incorporation of [2- C]DX and [2- C]MVL into GA Feeding experiments were carried out in MS liquid medium supplemented with 1% sucrose (total 15 days of incubation). GA was analyzed by GC-MS as a methylester derivative. KRI, Kovats retention index. Mass spectrum KRI % Incorporation Molecular ion Fragment ion % relative intensity GA methyl ester Standard 2389 360 (5) 361 (1) 300 (100) 301 (24) 302 (3) 13 c [2- C]DX/cla1–1 2389 364 (2) 300 (5) 301 (12) 302 (17) 303 (34) 304 (100) 305 (28) 88 13 d [2- C]MVL/mevastatin 2389 360 (3) 361 (2) 300 (100) 301 (38) 302 (14) 303 (4) 7 The intensity of base ion peak was set as 100%. b 13 Incorporation rate was calculated from fragment ion peaks at m/z 300 –305 after the subtraction of natural C abundance. c 13 The cla1–1 seedlings were fed with 0.8 mM [2- C]DX d 13 The wild-type seedlings were fed with 3 mM [2- C]MVL in the presence of mevastatin. TABLE III thesis of GAs, we analyzed GA from the [2- C]DX-treated 13 13 Incorporation of [2- C]DX and [2- C]MVL at different concentrations cla11 plants by GC-MS. GA is the first GA in the GA into ent-kaurene and campesterol biosynthesis pathway and is converted to biologically active Feeding experiments were carried out in the MS liquid medium forms by several oxidation steps. Because GAs accumulate to supplemented with 1% sucrose (total 15 days of incubation). very low levels in plants and this analysis required much more 13 a 13 b [2- C]DX (mM) [2- C]MVL (mM) tissue, we set up new liquid culture conditions to grow the 0.8 0.4 0.2 3 1 plants on a larger scale. In the new system, we reduced the % incorporation sucrose concentration to 1% (formerly 3%) to reduce the chance ent-Kaurene 87 81 68 5 8 of fungal contamination. 13 Campesterol 7 4 3 80 80 The mass spectrum of GA methyl ester from [2- C]DX-fed 13 a 13 [2- C]DX was fed to cla1–1 seedlings. cla11 seedlings demonstrated the incorporation of four C b 13 [2- C]MVL was fed to wild-type seedlings in the presence of labels per molecule, illustrating the contribution of the MEP mevastatin. pathway to its biosynthesis. The incorporation of [2- C]DX into GA (88%), which was determined after subtraction of the 13 13 natural C abundance, was similar to that of [2- C]DX into mechanism for crossover between MEP and MVA pathways, we ent-kaurene (87%) at the same concentration of the substrate determined the incorporation of labeled precursors when the (Tables II and III). To examine the role of the MVA pathway in metabolic flux through one of the pathways is limited. This set GA biosynthesis, another large scale culture was carried out to of experiments was carried out in liquid culture containing 1% test the incorporation of [2- C]MVL in the presence of mevas- sucrose. tatin. GC-MS analysis showed that 7 and 5% of the isoprene The cla11 albino phenotype was rescued only partially by units of GA and ent-kaurene, respectively, came from 0.2 and 0.4 mM DX (Fig. 2A). This suggests that DX is still [2- C]MVL (Tables II and III), indicating a minor contribution limiting, and therefore, the MEP pathway is not saturated. of the MVA pathway to the synthesis of GA through Under these conditions, [2- C]DX was still incorporated into ent-kaurene. campesterol, although the incorporation was lower (3 and 4% Effects of Exogenous DX and MVA on the Growth of cla1–1or at 0.2 and 0.4 mM, respectively; Table III) than that obtained Mevastatin-treated Wild-type Seedlings—Our feeding experi- with the higher concentration of [2- C]DX (7%). Similarly, ments showed a minor role of the MVA pathway in ent-kaurene [2- C]MVL was incorporated into ent-kaurene (8%; Table III) synthesis. To examine how this minor incorporation affects when mevastatin-treated plants were fed with 1 mM MVL, plant growth, we analyzed the effect of exogenous MVL on the which did not fully rescue the growth inhibition by mevastatin phenotype of cla11 seedlings. Fig. 2C shows that cla11 (Fig. 2B). These results indicate that the incorporation of 13 13 seedlings accumulate green-yellow pigments in the presence of [2- C]MVL into ent-kaurene and that of [2- C]DX into 1–3mM MVL. However, the cla11 albino phenotype was not campesterol occurs even when the MVA and MEP pathways, fully rescued even at higher doses of MVL (data not shown). respectively, are limiting. Our data also showed that the con- These results support our conclusion that the MVA pathway centrations of C-labeled precursors in the media can modu- plays a minor role in the isoprenoid biosynthesis in plastids, late the amount of C incorporation into the products, which is where the major route is the MEP pathway. Likewise, we evident by the decreased incorporation of [2- C]DX into ent- treated wild-type seedlings with varying concentrations of DX kaurene at 0.2 mM [2- C]DX (68%) relative to that observed at in the presence of mevastatin. Exogenous DX at 0.5 and 1 mM 0.8 mM [2- C]DX (87%) (Table III). partially rescued the growth defect caused by mevastatin (Fig. DISCUSSION 2D). However, the growth inhibition was not completely re- stored even at higher concentrations of DX in the media. These Our feeding experiments using C-labeled precursors have results are consistent with our GC-MS data showing that the demonstrated that both MEP and MVA pathways can supply MEP pathway can partially contribute to cytosolic sterol precursors for the biosynthesis of GAs in Arabidopsis seedlings. biosynthesis. This study provides the first evidence that GAs can be synthe- Crossover Between the Two Pathways at a Lower Dose of sized through the MEP pathway. Because GAs and their pre- 13 13 [2- C]DX or [2- C]MVL—Using feeding experiments with cursor ent-kaurene are present at extremely low levels in plant 13 13 [2- C]DX or [2- C]MVL, we showed that either precursor can tissues, we needed a sensitive method to monitor the incorpo- be incorporated into both ent-kaurene and campesterol with ration of isotopically labeled intermediates. The use of mevas- different efficiencies. These experiments were carried out at tatin and the cla11 mutation to block individual isoprenoid 13 13 concentrations of exogenous [2- C]DX or [2- C]MVL that pathways allowed us to label metabolites efficiently. In stevio- were sufficient to overcome the effect of the cla11 mutation or side biosynthesis in S. rebaudiana, feeding experiments with mevastatin, respectively, nearly completely. To investigate the [1- C]glucose and NMR were used to show that the diterpene Contribution of the MVA and MEP Pathways to GA Biosynthesis 45193 moiety (a derivative of ent-kaurenoic acid) is synthesized needs to be determined. An active uptake of IPP into isolated through the MEP pathway. A contribution from the MVA path- plastids in a facilitated-diffusion manner (43– 45) implies that way was not evident in this study. The inconsistency between IPP may be involved in the precursor exchange between the this study and ours may be because of differences in plant MVA and MEP pathways. It is interesting to note that the cla11 phenotype was species, tissue, or development stage, roles of the products (secondary metabolite versus growth regulator), or the inability rescued only partially even at a high dose of MVL (Fig. 2C). This demonstrates that the MEP pathway is still required for to detect minor C incorporation by NMR. Because glucose can normal plant growth even when the flux of MVA pathway is be incorporated into both the MEP and MVA pathways, C-labeled glucose has often been used to label isoprenoids in elevated. Taken together, our current data suggest that the uptake of an intermediate from the MVA pathway into the vivo (38, 39). However, it is not known whether exogenously isoprenoid pathway in plastids does occur, but that this mech- applied C-glucose is equally distributed to the two pathways. anism is not sufficient to fully complement the defect in the This would be a crucial question when both MEP and MVA MEP pathway. This also appears to be true for the other direc- pathways are involved in the biosynthesis of target compounds. tion, uptake from the MEP pathway to the MVA pathway. We Crossover between the MEP and MVA pathways has been noted that the incorporation of labeled precursors into ent- demonstrated in the biosynthesis of several terpenoids in other kaurene and campesterol was greater in our first set of exper- plant species using isotopically labeled DX or MVL (2, 40 – 42). iments performed in 3% sucrose than in the second set in 1% However, in all cases, cellular concentrations of the precursor sucrose. This observation suggests that sucrose concentrations appeared to be elevated because the labeled compounds were may affect the uptake of C-labeled substrates by plants fed to plants without reducing the endogenous levels of these and/or the status of two isoprenoid pathways. precursors. By inhibiting each isoprenoid pathway and moni- In summary, our study showed that both isoprenoid path- toring the phenotypic changes at different doses of the labeled ways are involved in the biosynthesis of GAs, which are precursors, we were able to estimate the status of isoprenoid growth-promoting hormones in plants. We predict that the pathways. This helped us to predict whether the feeding exper- relative contribution of each pathway may be modulated dur- iments were done under physiologically relevant conditions. ing plant development, by environmental cues and by the sta- Our results showed that the concentrations of [2- C]DX in the tus of the other pathway. Molecular genetic approaches in the media can greatly affect the ratio of C labels in the products model species Arabidopsis would be useful to uncover how each (Table III). Thus, the C incorporation determined in different isoprenoid pathway is regulated and whether the crossover feeding experiments must be carefully interpreted. When the plays any role in controlling isoprenoid biosynthesis in cyto- MEP pathway is limiting in the cla11 seedlings at 0.2 mM DX plasm and plastids. (judged by the incomplete recovery of the cla11 phenotype; Fig. 2A), the incorporation of [2- C]DX into ent-kaurene was Acknowledgments—We thank Dr. Shozo Fujioka (RIKEN) for advise only 68%, whereas it increased to 87% at a higher [2- C]DX on feeding experiments, Dr. Patricia Leo ´ n (National Autonomous Uni- versity of Mexico, Mexico) for providing the cla11 seeds. We also concentration. It is unclear at present whether the reduced thank Drs. Peter Hedden (University of Bristol, Bristol, UK), Hiroshi labeling ratio at the lower dose of [2- C]DX is due to the leaky Kawaide (Tokyo University of Agriculture and Technology, Tokyo), nature of the cla11 mutant (e.g. presence of isozymes) or to Kazunori Okada (Tokyo Gakugei University, Tokyo), Juan M. Este ´ vez the crossover from the MVA pathway. These observations in- (RIKEN), Doris Albinsky (RIKEN), and Eiji Nambara (RIKEN) for dicate that the amount of C incorporation into the product helpful comments on the manuscript. does not necessarily reflect the relative contribution of each REFERENCES pathway under normal growth conditions. These results also 1. Sacchettini, J. C., and Poulter, C. D. (1997) Science 277, 1788 –1789 suggest that the relative contribution of each isoprenoid path- 2. Rohmer, M. (1999) Nat. Prod. Rep. 16, 565–574 way could vary when either pathway is up- or down-regulated 3. Lichtenthaler, H. K. (1999) Annu. Rev. Plant Physiol. Plant Mol. 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Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Gibberellins inArabidopsis

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 47, Issue of November 22, pp. 45188 –45194, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Gibberellins in Arabidopsis* Received for publication, August 23, 2002, and in revised form, September 12, 2002 Published, JBC Papers in Press, September 12, 2002, DOI 10.1074/jbc.M208659200 Hiroyuki Kasahara‡, Atsushi Hanada‡, Tomohisa Kuzuyama§, Motoki Takagi§, Yuji Kamiya‡ and Shinjiro Yamaguchi‡ From the ‡Laboratory for Cellular Growth and Development, Growth Physiology Research Group, Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama 351-0198, Japan and §Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan already implicated in the 1960s from feeding experiments with Gibberellins (GAs) are diterpene plant hormones es- 14 14 sential for many developmental processes. Although the CO and [ C]MVA, which showed that these substrates GA biosynthesis pathway has been well studied, our labeled distinct groups of terpenoids (5). The MEP pathway knowledge on its early stage is still limited. There are was first described for eubacteria (6), and enzymes catalyzing two possible routes for the biosynthesis of isoprenoids the MEP pathway have been identified mainly in Escherichia leading to GAs, the mevalonate (MVA) pathway in the coli (7, 8). Although the precise reactions in the late steps of cytosol and the methylerythritol phosphate (MEP) path- MEP pathway have still to be determined, the overall pathway way in plastids. To distinguish these possibilities, in E. coli has recently been proposed (9). Because orthologs of metabolites from each isoprenoid pathway were selec- each of the bacterial genes of the MEP pathway are present in tively labeled with Cin Arabidopsis seedlings. Arabidopsis thaliana (8, 10), the same set of enzymes are likely Efficient C-labeling was achieved by blocking the to be involved in the MEP pathway in plants. endogenous pathway chemically or genetically during Gibberellins (GAs) are a class of plant hormones essential for the feed of a C-labeled precursor specific to the MVA or many aspects of plant growth and development, such as seed MEP pathways. Gas chromatography-mass spectrome- germination, stem elongation, and flower development (11, 12). try analyses demonstrated that both MVA and MEP The GA biosynthesis pathway in higher plants has been stud- pathways can contribute to the biosyntheses of GAs and ied in detail in cell-free systems from immature seeds of campesterol, a cytosolic sterol, in Arabidopsis seedlings. Cucurbita maxima, Pisum sativum, and Phaseolus vulgaris While GAs are predominantly synthesized through the (13). Because C-labeled MVA was efficiently incorporated MEP pathway, the MVA pathway plays a major role in into ent-kaurene (a GA precursor) in these cell-free systems, it the biosynthesis of campesterol. Consistent with some has long been assumed that GAs are derived from MVA in crossover between the two pathways, phenotypic de- fects caused by the block of the MVA and MEP pathways plants. The GA biosynthesis pathway has also been studied were partially rescued by exogenous application of the extensively in a GA-producing fungus Gibberella fujikuroi (14). MEP and MVA precursors, respectively. We also provide The incorporation of labeled MVA into GAs in cultured mycelia evidence to suggest that the MVA pathway still contrib- of this fungus also supported the premise that GAs are formed utes to GA biosynthesis when this pathway is limiting. from MVA in this organism (15). Several lines of evidence from recent work have indicated that ent-kaurene is synthesized in the plastids of plants (16 – Isoprenoids comprise a broad range of natural products that 19). Therefore, the MEP pathway in plastids may play a role in are synthesized by the condensation of the two precursors, providing IPP and DMAPP for ent-kaurene biosynthesis (Fig. isopentenyl diphosphate (IPP) and dimethylallyl diphosphate 1). There is some indirect evidence to support this hypothesis. (DMAPP) (1). Plants have two distinct biosynthetic routes for Antisense suppression of genes encoding enzymes in the Ara- the formation of IPP and DMAPP, the mevalonate (MVA) path- bidopsis MEP pathway resulted in elevated expression of a way and the newly discovered methylerythritol phosphate GA-down-regulated gene, GA4 (20), and reduced production of (MEP) pathway (Fig. 1) (2, 3). In plants, the MVA pathway ent-kaurene (21). Recently, the biosynthetic origin of stevio- plays an essential role in the biosynthesis of sterols and ses- side, of which the aglycone is a derivative of ent-kaurenoic acid, quiterpenoids in the cytoplasm (4). On the other hand, the MEP has been studied in Stevia rebaudiana leaves (22), where this pathway is generally responsible for the formation of carote- diterpene glycoside accumulates to more than 10% of the leaf noids, mono- and di-terpenoids, plastoquinones, and the prenyl dry weight as a secondary metabolite. This high abundance 13 13 group of chlorophylls in plastids (3). The occurrence of two allowed the C-labeling pattern from [1- C]glucose in steviol separate isoprenoid biosynthesis pathways in plants was to be determined by NMR and indicated that steviosides are synthesized through the MEP pathway. However, this method * The costs of publication of this article were defrayed in part by the is not feasible for GAs due to their low abundance in plant payment of page charges. This article must therefore be hereby marked tissues. Thus, to determine whether GAs are synthesized “advertisement” in accordance with 18 U.S.C. Section 1734 solely to through the MEP pathway in general, a more sensitive system indicate this fact. for detecting the labeled products is required. To whom correspondence should be addressed. Tel.: 81-48-467- 9552; Fax: 81-48-462-4691; E-mail: [email protected]. To this end, we used an Arabidopsis albino mutant cla11, The abbreviations used are: IPP, isopentenyl diphosphate; GA, gib- which is defective in 1-deoxy-D-xylulose 5-phosphate synthase berellin; DMAPP, dimethylallyl diphosphate; DX, 1-deoxy-D-xylulose; in the MEP pathway (23). The cla11 phenotype can be res- MEP, methylerythritol phosphate; MVA, mevalonate; MVL, mevalono- cued almost completely by treatment with exogenous 1-deoxy- lactone; TMS, tetramethylsilane; GC-MS, gas chromatography-mass spectrometry; MS medium, Murashige and Skoog medium. D-xylulose (DX), which is converted to a MEP pathway inter- 45188 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Contribution of the MVA and MEP Pathways to GA Biosynthesis 45189 FIG.1. The two possible isoprenoid biosynthesis pathways leading to GAs in plants. A cell-free system from C. maxima endosperm can convert MVA to ent-kaurene and GAs. ent-Kaurene is pro- duced in plastids and then converted to GAs by following oxidation reactions in cytosol. Crossover of common isoprenoid precursors (IPP, geranylgeranyl diphos- phate (GGPP), or farnesyl diphosphate (FPP)) between cytosol and plastids has been suggested in several plant species. Dashed arrows indicate multiple biosyn- thetic steps. HMGR, 3-hydroxy-3-meth- ylglutaryl-CoA reductase; DXS, 1-deoxy- D-xylulose 5-phosphate synthase; DXP, 1-deoxy-D-xylulose 5-phosphate; DXR, 1-deoxy-D-xylulose 5-phosphate reduc- toisomerase; GAP, glyceraldehyde 3-phosphate. concentration 1 M) was added, and then the plants were grown for an mediate 1-deoxy-D-xylulose 5-phosphate in plants. This system additional 3 days before analyzing ent-kaurene. GA and campesterol allowed us to label the products from the MEP pathway effi- were analyzed without uniconazole treatment. ciently in vivo using [2- C]DX. To evaluate the role of the Feeding of [2- C]MVL to Mevastatin-treated Plants—Wild-type cytosolic MVA pathway in GA biosynthesis in the same system, seedlings were grown for 5 days on MS agar media before transferring C-labeled mevalonolactone (MVL) was fed to plants that were to MS liquid media. Immediately after the transfer, mevastatin (MeOH treated with mevastatin, an inhibitor of the MVA pathway. solution, final concentration 10 M) and [2- C]MVL (filter-sterilized H O solution) were added aseptically to the liquid media, and the plants Our gas chromatography-mass spectrometry (GC-MS) analysis 2 were grown for 9 days. ent-Kaurene, GA , and campesterol were ana- demonstrated that GAs are predominantly synthesized from lyzed as described above. the MEP pathway in Arabidopsis seedlings. However, our GC-MS Analysis of ent-Kaurene, GA , and Campesterol—For ent- results also indicated a minor contribution of the MVA pathway kaurene analyses, seedlings (3 g) were pulverized with a mortar and to GA biosynthesis. Cooperation of both isoprenoid pathways pestle chilled by liquid N . Powdered tissues were extracted with 80% was also evident for the biosynthesis of the sterol campesterol MeOH (25 ml) overnight. The 80% MeOH extract was then partitioned against n-hexane (15 ml) three times, and the combined n-hexane (a precursor for brassinosteroids), which is formed in the fraction was evaporated to 1 ml. The n-hexane fraction was subjected to cytosol. SiO gel column chromatography (column size, 5  2 cm) and eluted with 15 ml of n-hexane. The eluate was carefully evaporated to 20 l EXPERIMENTAL PROCEDURES under gentle N flow and analyzed by GC-MS. GC-MS analysis was Plant Materials and Growth Conditions—A. thaliana ecotype Was- performed on a GC-mate II mass spectrometer (JEOL, Tokyo, Japan) silewskija (WS) was used in this study. Plants were grown at 21 °C connected to a Agilent 6890 series GC system with a 30-m  0.25-mm using a 16-h light/8-h dark photoperiod with cool-white illumination. capillary column DB-5 MS (0.25-m film thickness, J & W Scientific). Wild-type (WS-2) and the cla11 mutant (24) were germinated and GA and campesterol were extracted from 2227-g and 0.4-g seedlings grown on Murashige and Skoog (MS) agar media (pH 5.7) supplemented and derivatized to methyl ester and TMS ether, respectively, and ana- 1 1 with thiamin hydrochloride (3 gml ), nicotinic acid (5 gml ), pyr- lyzed as reported previously (29, 30). ent-Kaurene and GA were iden- idoxin hydrochloride (0.5 gml ), and 1 or 3% (w/v) sucrose. Liquid tified by Kovats retention indices (31), and full mass spectra obtained culture was carried out in 15 ml of MS media in 100-ml flasks on a by GC-MS (29, 32). shaker (100 rpm). Chemicals—DX and [2- C]DX (95% labeled) were synthesized using RESULTS pyruvate or [2- C]pyruvate (99% labeled, Aldrich) as previously re- 13 13 13 ported (25). To determine the C-labeling ratio of [2- C]DX, DX and Incorporation of [2- C]DX into ent-Kaurene—The Arabidop- [2- C]DX (ca. 100 ng) were converted to trimethylsilyl derivatives by sis cla11 mutant is defective in 1-deoxy-D-xylulose 5-phos- heating at 70 °C with N,O-bis(trimethylsilyl)acetamide  trimethyl- phate synthase in the MEP pathway and displays a seedling- chlorosilane  trimethylsilylimidazole (3:2:3, 50 l, Supelco) and pyri- lethal albino phenotype (23, 24). Previous studies show that the dine (50 l) for 20 min and then analyzed by GC-MS as previously 13 cla11 phenotype is in part restored when grown on agar reported (26). DL-MVL and [2- C]MVL (99% labeled) were purchased media containing DX (23). We found that the cla11 phenotype from Aldrich, and mevastatin was from Sigma. ent-[1,7,12,18- C ] Kaurene was produced from [2- C]MVA by a cell-free system prepared can be better rescued in liquid culture than on agar media in from C. maxima endosperm as previously reported (27, 28). the presence of DX, possibly because of better uptake of the Feeding of [2- C]DX to the cla1-1 Mutant—The albino cla11 ho- chemical by seedlings. Fig. 2A shows that, in the presence of mozygotes were selected from the progeny of CLA1/cla11 plants after 0.8 –1.0 mM DX, the phenotype of the cla11 plants was re- incubation on MS agar media for 9 days. The cla11 seedlings were 13 stored to that of the wild type. To label isoprenoids that are then transferred to MS liquid media. [2- C]DX was dissolved in H O, produced via the MEP pathway in vivo, the cla11 mutant was filter-sterilized, and added aseptically to the liquid culture. Twelve days after the transfer to liquid media, uniconazole (EtOH solution, final treated with 1 mM [2- C]DX for 15 days. Again, the rescue of 45190 Contribution of the MVA and MEP Pathways to GA Biosynthesis FIG.2. A–D, effect of DX and MVL on Arabidopsis cla11 and mevastatin-treated wild-type seedlings. Numbers indicate concentra- tions (mM)ofDX(in A and D) or MVL (in B and C). All experiments were performed in MS liquid media supplemented with 1% sucrose. A, restoration of cla11 seedlings by DX. The albino cla11 seedlings (9 days after sowing) were incubated in MS liquid media for 10 days with DX. For wild-type, see Control in B. B, restoration of mevastatin-treated wild-type seedlings by MVL. WT seedlings (5 days after sowing) were incubated for 7 days in MS liquid media with MVL in the presence of mevastatin (10 M). Control indicates the plant incubated without mevastatin and MVL. C, effect of MVL on cla11 seedlings. The albino cla11 seedlings (5 days after sowing) were incubated in MS liquid media for 3 days with MVL. D, effect of DX on mevastatin-treated wild-type seedlings. WT seedlings (5 days after sowing) were incubated for 5 days in MS liquid media with DX in the presence of mevastatin (10 M). Control indicates the plant incubated without mevastatin and DX. the albino phenotype nearly to wild-type confirmed that addition to the molecular ion at m/z 272, four isotope peaks at [2- C]DX was metabolized as required. m/z 273, 274, 275, and 276 were observed, indicating that 1– 4 To examine the role of the MEP pathway in GA biosynthesis, C labels were introduced into ent-kaurene (Table I). Subtrac- 13 13 13 we first determined the incorporation of [2- C]DX into ent- tion of natural C abundance indicated that [2- C]MVL pro- kaurene, a tetracyclic hydrocarbon precursor for all GAs, by vided 53% of the isoprene units to ent-kaurene under this GC-MS. Because ent-kaurene accumulates at low levels in Ara- condition. As discussed above, the mass spectrum of ent-kau- bidopsis seedlings (data not shown), plants were treated with 1 rene produced from [2- C]MVA through the MVA pathway M uniconazole for 3 days to block ent-kaurene metabolism contains a fragment ion at m/z 233 ([M-43] ), because ring D of before GC-MS analysis (21). If ent-kaurene is synthesized from ent-kaurene would not be labeled with C. Considering the 13 13 13 [2- C]DX through the MEP pathway, four C atoms would be amount of C incorporation (53%) calculated from the molec- introduced at the positions shown in Fig. 3 (Route 2). Consist- ular ion cluster, the relative intensity of the fragment ion at ent with this prediction, the mass spectrum of ent-kaurene m/z 233 confirms the incorporation of [2- C]MVL into ent- from the [2- C]DX-treated cla11 plants indicated a peak at kaurene through the MVA pathway. These results illustrate m/z 276, which corresponds to the molecular ion with four C that the MVA pathway also contributes to the biosynthesis of atoms per molecule (Table I). The peak at m/z 229 [M-43] of ent-kaurene in Arabidopsis seedlings. 13 13 non-labeled ent-kaurene is a fragment ion after loss of ring D Incorporation of [2- C]DX and [2- C]MVL into Campes- (C H and three hydrogen atoms) (Fig. 3), as previously dem- terol—Our feeding experiments showed that both the MEP and 3 4 onstrated by deuterium-labeling experiments (32). Impor- MVA pathways can provide precursors for the biosynthesis of tantly, the corresponding ion peak from the [2- C]DX-treat- ent-kaurene. To study the contributions of these two pathways ed plants was observed at m/z 232 ([M-44] ). This indicates to cytosolic sterol biosynthesis in Arabidopsis seedlings, the 13 13 13 the loss of one C label in ring D and is consistent with the incorporation of [2- C]DX and [2- C]MVL into campesterol predicted labeling pattern through the MEP pathway from was analyzed by GC-MS. [2- C]DX. To confirm this result, we analyzed ent-kaurene The mass spectrum of campesterol-TMS from the 13 13 produced from [2- C]MVA by a cell-free system from C. [2- C]MVL-fed seedlings showed that 98% of its isoprene units maxima endosperm (27, 28), where ring D would not contain was C-labeled (Table I). A peak at m/z 477, which corresponds 13 13 13 C-labels (Fig. 3, Route 3). GC-MS showed that four C to the molecular ion with five C atoms per molecule, is in atoms per molecule were incorporated into ent-kaurene and agreement with the expected labeling pattern because one of that the corresponding fragment ion was at m/z 233 ([M- the six C-labels introduced in the precursor squalene will be 43] ). Subtraction of natural C abundance revealed that eliminated by C-4 demethylation during campesterol biosyn- 99% of the C building blocks had been derived from thesis (Fig. 3, Route 4). The fragment ion peak at m/z 343 [2- C]DX through the MEP pathway in the cla11 seedling ([M-129] ) of non-labeled campesterol-TMS is attributed to loss (33). of C-1, -2, and -3 and a TMS-ether group of the A-ring (35), as Incorporation of [2- C]MVL into ent-Kaurene—Mevastatin shown in Fig. 3. The corresponding fragment ion from the inhibits 3-hydroxy-3-methylglutaryl-CoA reductase in the [2- C]MVL-fed seedlings at m/z 347 ([M-130] ), which indi- MVA pathway and causes severe growth inhibition, which is cates loss of one C-label, is consistent with the predicted restored by simultaneous application of MVL. To study the labeling pattern (Fig. 3). involvement of the MVA pathway in GA biosynthesis, To examine the role of MEP pathway in cytosolic sterol 13 13 [2- C]MVL was fed to Arabidopsis seedlings that were incu- synthesis, campesterol was analyzed in the [2- C]DX-treated bated with mevastatin to label MVA-derived isoprenoids (34). In cla11 seedlings by GC-MS. Campesterol can be labeled with 13 13 our liquid culture conditions, 10 M mevastatin was effective in C at six positions when [3- C]IPP, originating from inhibiting seedling growth, and this inhibitory effect was nearly [2- C]DX, is incorporated (Fig. 3, Route 1), in contrast to the 13 13 completely abolished by the addition of 3 mM MVL (Fig. 2B). five C atoms incorporated from [2- C]MVL. The mass spec- ent-Kaurene was analyzed by GC-MS from seedlings that trum of campesterol-TMS from [2- C]DX-fed cla11 seed- were treated with 10 M mevastatin and 3 mM [2- C]MVL. In lings showed a peak at m/z 478 (Table I). This indicated that Contribution of the MVA and MEP Pathways to GA Biosynthesis 45191 13 13 FIG.3. Predicted labeling patterns of ent-kaurene, GA and campesterol with [2- C]DX or [2- C]MVL. Red arrows indicate possible 13 13 metabolic conversion of [2- C]DX through the MEP pathway, and blue arrows are those of [2- C]MVL through the MVA pathway. Red stars 13 13 13 specify positions of C atoms from [2- C]DX, and blue stars specify those from [2- C]MVL. The ring D of ent-kaurene is shown in orange, the A-ring of campesterol was shown in pink. , derivatized from campesterol by trimethylsilylation with N-methyl-N-trimethylsilyltrifluoroacetamide. , derivatized from GA by methylation using diazomethane. DXP, 1-deoxy-D-xylulose 5-phosphate; GGPP, geranylgeranyl diphosphate; or FPP, farnesyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A. TABLE I GC-MS data for ent-kaurene and campesterol-TMS Feeding experiments were carried out in MS liquid media supplemented with 3% sucrose (total 15 days of incubation). KRI, Kovats retention index. Mass spectrum KRI % Incorporation Molecular ion Fragment ion % relative intensity ent-Kaurene Standard 2082 272 (100) 273 (21) 274 (3) 229 (93) 230 (18) 231 (4) 13 b [2- C]DX/cla1–1 2082 275 (25) 276 (98) 277 (21) 232 (100) 233 (22) 234 (3) 99 13 c [2- C]MVL/mevastatin 2082 272 (70) 273 (48) 274 (52) 27 (99) 229 (78) 230 (64) 231 (78) 232 (100) 53 276 (71) 233 (49) [2- C]MVA/C. maxima 2082 276 (100) 277 (15) 232 (39) 233 (100) Campesterol TMS Standard 472 (42) 473 (16) 474 (4) 343 (100) 344 (27) 345 (4) 13 b [2- C]DX/cla1–1 472 (63) 473 (65) 474 (42) 47 (23) 343 (88) 344 (100) 345 (89) 346 (60) 27 476 (15) 477 (13) 478 (13) 347 (40) 348 (34) 349 (33) 350 (7) 13 c [2- C]MVL/mevastatin 476 (6) 477 (38) 478 (14) 345 (12) 346 (43) 347 (100) 348 (26) 98 The intensity of base ion peak was set as 100%. b 13 The cla1–1 seedlings were fed with 1 mM [2- C]DX. c 13 The wild-type seedlings were fed with 3 mM [2- C]MVL in the presence of mevastatin. a maximum of six C atoms were incorporated per molecule, MVA pathway also contributes to the biosynthesis of campes- which is consistent with the incorporation of IPP/DMAPP terol in Arabidopsis seedlings. 13 13 13 derived from [2- C]DX into cytosolic campesterol biosynthe- Incorporation of [2- C]DX and [2- C]MVL into GA —Al- sis. Furthermore, considering the amount of C incorpora- though ent-kaurene is a common intermediate for all GAs, it is tion (27%) estimated from the molecular ion cluster, the also known to serve as a precursor for other diterpenoids such relative abundance of the fragment ion at m/z 349 indicates as stevioside in S. rebaudiana (22) and kaurenolides in C. no C label at C-1 of the A-ring. This observation agrees with maxima (36, 37). In Arabidopsis, it has not been established the expected labeling pattern through the MEP pathway whether ent-kaurene serves as a precursor solely for GAs. To from [2- C]DX (Route 1). These results indicate that the determine conclusively the role of MEP pathway in the biosyn- 45192 Contribution of the MVA and MEP Pathways to GA Biosynthesis TABLE II 13 13 Incorporation of [2- C]DX and [2- C]MVL into GA Feeding experiments were carried out in MS liquid medium supplemented with 1% sucrose (total 15 days of incubation). GA was analyzed by GC-MS as a methylester derivative. KRI, Kovats retention index. Mass spectrum KRI % Incorporation Molecular ion Fragment ion % relative intensity GA methyl ester Standard 2389 360 (5) 361 (1) 300 (100) 301 (24) 302 (3) 13 c [2- C]DX/cla1–1 2389 364 (2) 300 (5) 301 (12) 302 (17) 303 (34) 304 (100) 305 (28) 88 13 d [2- C]MVL/mevastatin 2389 360 (3) 361 (2) 300 (100) 301 (38) 302 (14) 303 (4) 7 The intensity of base ion peak was set as 100%. b 13 Incorporation rate was calculated from fragment ion peaks at m/z 300 –305 after the subtraction of natural C abundance. c 13 The cla1–1 seedlings were fed with 0.8 mM [2- C]DX d 13 The wild-type seedlings were fed with 3 mM [2- C]MVL in the presence of mevastatin. TABLE III thesis of GAs, we analyzed GA from the [2- C]DX-treated 13 13 Incorporation of [2- C]DX and [2- C]MVL at different concentrations cla11 plants by GC-MS. GA is the first GA in the GA into ent-kaurene and campesterol biosynthesis pathway and is converted to biologically active Feeding experiments were carried out in the MS liquid medium forms by several oxidation steps. Because GAs accumulate to supplemented with 1% sucrose (total 15 days of incubation). very low levels in plants and this analysis required much more 13 a 13 b [2- C]DX (mM) [2- C]MVL (mM) tissue, we set up new liquid culture conditions to grow the 0.8 0.4 0.2 3 1 plants on a larger scale. In the new system, we reduced the % incorporation sucrose concentration to 1% (formerly 3%) to reduce the chance ent-Kaurene 87 81 68 5 8 of fungal contamination. 13 Campesterol 7 4 3 80 80 The mass spectrum of GA methyl ester from [2- C]DX-fed 13 a 13 [2- C]DX was fed to cla1–1 seedlings. cla11 seedlings demonstrated the incorporation of four C b 13 [2- C]MVL was fed to wild-type seedlings in the presence of labels per molecule, illustrating the contribution of the MEP mevastatin. pathway to its biosynthesis. The incorporation of [2- C]DX into GA (88%), which was determined after subtraction of the 13 13 natural C abundance, was similar to that of [2- C]DX into mechanism for crossover between MEP and MVA pathways, we ent-kaurene (87%) at the same concentration of the substrate determined the incorporation of labeled precursors when the (Tables II and III). To examine the role of the MVA pathway in metabolic flux through one of the pathways is limited. This set GA biosynthesis, another large scale culture was carried out to of experiments was carried out in liquid culture containing 1% test the incorporation of [2- C]MVL in the presence of mevas- sucrose. tatin. GC-MS analysis showed that 7 and 5% of the isoprene The cla11 albino phenotype was rescued only partially by units of GA and ent-kaurene, respectively, came from 0.2 and 0.4 mM DX (Fig. 2A). This suggests that DX is still [2- C]MVL (Tables II and III), indicating a minor contribution limiting, and therefore, the MEP pathway is not saturated. of the MVA pathway to the synthesis of GA through Under these conditions, [2- C]DX was still incorporated into ent-kaurene. campesterol, although the incorporation was lower (3 and 4% Effects of Exogenous DX and MVA on the Growth of cla1–1or at 0.2 and 0.4 mM, respectively; Table III) than that obtained Mevastatin-treated Wild-type Seedlings—Our feeding experi- with the higher concentration of [2- C]DX (7%). Similarly, ments showed a minor role of the MVA pathway in ent-kaurene [2- C]MVL was incorporated into ent-kaurene (8%; Table III) synthesis. To examine how this minor incorporation affects when mevastatin-treated plants were fed with 1 mM MVL, plant growth, we analyzed the effect of exogenous MVL on the which did not fully rescue the growth inhibition by mevastatin phenotype of cla11 seedlings. Fig. 2C shows that cla11 (Fig. 2B). These results indicate that the incorporation of 13 13 seedlings accumulate green-yellow pigments in the presence of [2- C]MVL into ent-kaurene and that of [2- C]DX into 1–3mM MVL. However, the cla11 albino phenotype was not campesterol occurs even when the MVA and MEP pathways, fully rescued even at higher doses of MVL (data not shown). respectively, are limiting. Our data also showed that the con- These results support our conclusion that the MVA pathway centrations of C-labeled precursors in the media can modu- plays a minor role in the isoprenoid biosynthesis in plastids, late the amount of C incorporation into the products, which is where the major route is the MEP pathway. Likewise, we evident by the decreased incorporation of [2- C]DX into ent- treated wild-type seedlings with varying concentrations of DX kaurene at 0.2 mM [2- C]DX (68%) relative to that observed at in the presence of mevastatin. Exogenous DX at 0.5 and 1 mM 0.8 mM [2- C]DX (87%) (Table III). partially rescued the growth defect caused by mevastatin (Fig. DISCUSSION 2D). However, the growth inhibition was not completely re- stored even at higher concentrations of DX in the media. These Our feeding experiments using C-labeled precursors have results are consistent with our GC-MS data showing that the demonstrated that both MEP and MVA pathways can supply MEP pathway can partially contribute to cytosolic sterol precursors for the biosynthesis of GAs in Arabidopsis seedlings. biosynthesis. This study provides the first evidence that GAs can be synthe- Crossover Between the Two Pathways at a Lower Dose of sized through the MEP pathway. Because GAs and their pre- 13 13 [2- C]DX or [2- C]MVL—Using feeding experiments with cursor ent-kaurene are present at extremely low levels in plant 13 13 [2- C]DX or [2- C]MVL, we showed that either precursor can tissues, we needed a sensitive method to monitor the incorpo- be incorporated into both ent-kaurene and campesterol with ration of isotopically labeled intermediates. The use of mevas- different efficiencies. These experiments were carried out at tatin and the cla11 mutation to block individual isoprenoid 13 13 concentrations of exogenous [2- C]DX or [2- C]MVL that pathways allowed us to label metabolites efficiently. In stevio- were sufficient to overcome the effect of the cla11 mutation or side biosynthesis in S. rebaudiana, feeding experiments with mevastatin, respectively, nearly completely. To investigate the [1- C]glucose and NMR were used to show that the diterpene Contribution of the MVA and MEP Pathways to GA Biosynthesis 45193 moiety (a derivative of ent-kaurenoic acid) is synthesized needs to be determined. An active uptake of IPP into isolated through the MEP pathway. A contribution from the MVA path- plastids in a facilitated-diffusion manner (43– 45) implies that way was not evident in this study. The inconsistency between IPP may be involved in the precursor exchange between the this study and ours may be because of differences in plant MVA and MEP pathways. It is interesting to note that the cla11 phenotype was species, tissue, or development stage, roles of the products (secondary metabolite versus growth regulator), or the inability rescued only partially even at a high dose of MVL (Fig. 2C). This demonstrates that the MEP pathway is still required for to detect minor C incorporation by NMR. Because glucose can normal plant growth even when the flux of MVA pathway is be incorporated into both the MEP and MVA pathways, C-labeled glucose has often been used to label isoprenoids in elevated. Taken together, our current data suggest that the uptake of an intermediate from the MVA pathway into the vivo (38, 39). However, it is not known whether exogenously isoprenoid pathway in plastids does occur, but that this mech- applied C-glucose is equally distributed to the two pathways. anism is not sufficient to fully complement the defect in the This would be a crucial question when both MEP and MVA MEP pathway. This also appears to be true for the other direc- pathways are involved in the biosynthesis of target compounds. tion, uptake from the MEP pathway to the MVA pathway. We Crossover between the MEP and MVA pathways has been noted that the incorporation of labeled precursors into ent- demonstrated in the biosynthesis of several terpenoids in other kaurene and campesterol was greater in our first set of exper- plant species using isotopically labeled DX or MVL (2, 40 – 42). iments performed in 3% sucrose than in the second set in 1% However, in all cases, cellular concentrations of the precursor sucrose. This observation suggests that sucrose concentrations appeared to be elevated because the labeled compounds were may affect the uptake of C-labeled substrates by plants fed to plants without reducing the endogenous levels of these and/or the status of two isoprenoid pathways. precursors. By inhibiting each isoprenoid pathway and moni- In summary, our study showed that both isoprenoid path- toring the phenotypic changes at different doses of the labeled ways are involved in the biosynthesis of GAs, which are precursors, we were able to estimate the status of isoprenoid growth-promoting hormones in plants. We predict that the pathways. This helped us to predict whether the feeding exper- relative contribution of each pathway may be modulated dur- iments were done under physiologically relevant conditions. ing plant development, by environmental cues and by the sta- Our results showed that the concentrations of [2- C]DX in the tus of the other pathway. Molecular genetic approaches in the media can greatly affect the ratio of C labels in the products model species Arabidopsis would be useful to uncover how each (Table III). Thus, the C incorporation determined in different isoprenoid pathway is regulated and whether the crossover feeding experiments must be carefully interpreted. When the plays any role in controlling isoprenoid biosynthesis in cyto- MEP pathway is limiting in the cla11 seedlings at 0.2 mM DX plasm and plastids. (judged by the incomplete recovery of the cla11 phenotype; Fig. 2A), the incorporation of [2- C]DX into ent-kaurene was Acknowledgments—We thank Dr. Shozo Fujioka (RIKEN) for advise only 68%, whereas it increased to 87% at a higher [2- C]DX on feeding experiments, Dr. Patricia Leo ´ n (National Autonomous Uni- versity of Mexico, Mexico) for providing the cla11 seeds. We also concentration. It is unclear at present whether the reduced thank Drs. Peter Hedden (University of Bristol, Bristol, UK), Hiroshi labeling ratio at the lower dose of [2- C]DX is due to the leaky Kawaide (Tokyo University of Agriculture and Technology, Tokyo), nature of the cla11 mutant (e.g. presence of isozymes) or to Kazunori Okada (Tokyo Gakugei University, Tokyo), Juan M. Este ´ vez the crossover from the MVA pathway. These observations in- (RIKEN), Doris Albinsky (RIKEN), and Eiji Nambara (RIKEN) for dicate that the amount of C incorporation into the product helpful comments on the manuscript. does not necessarily reflect the relative contribution of each REFERENCES pathway under normal growth conditions. These results also 1. Sacchettini, J. C., and Poulter, C. D. (1997) Science 277, 1788 –1789 suggest that the relative contribution of each isoprenoid path- 2. Rohmer, M. (1999) Nat. Prod. Rep. 16, 565–574 way could vary when either pathway is up- or down-regulated 3. Lichtenthaler, H. K. (1999) Annu. Rev. Plant Physiol. Plant Mol. 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